Immobilized cholinesterase enzyme preparations and a process for the preparation thereof

ABSTRACT

Immobilized cholinesterase enzyme preparations are prepared by treating a polymeric resin, built up from acrylic acid and/or methacrylic acid and acryl amide and/or methacryl amide monomers with an acryl or allyl type cross-linking agent and containing at least 0.1 meq/g of --COOH functional groups, with a carbodiimide derivative which is soluble in water or is soluble in an organic solvent at temperatures below 0° C., applying a solution of cholinesterase enzyme with a pH of 4.5 to 8.5 to the resulting activated support, washing the resulting product, and drying it if desired.

The invention relates to new immobilized cholinesterase enzymepreparations as well as to a process for the preparation thereof.

It is known that cholinesterase enzyme, isolated most frequently fromhorse serum, can be applied to detect neurotoxins of phosphate ester,carbamate and sulfate ester type, and to determine the quantity ofneurotoxins occurring in air or water. The basis of detection andmeasurement is the inhibiting effect exerted on the enzyme byneurotoxins. Thus, immobilized cholinesterase enzyme preparations mayplay the central role of continuously operating measuring systems. Theprinciples of such measuring systems are described, among others, inAnal. Chem. 37, 1378 (1965) and Anal. Biochem. 51, 362 (1973).

Several methods have been described in the literature for theimmobilization of cholinesterase isolated from horse serum. According toAnal. Chem. 37, 1378 (1965), Anal. Biochem. 19, 587 (1967), Anal.Biochem. 33, 341 (1970) and U.S. Pat. No. 3,223,593 the enzyme isentrapped in various gels, primarily starch gels. U.S. Pat. No.3,223,593 also discloses the entrapment of the enzyme in agar andcarrageen gels. The use of polyacrylamide as entrapping medium issuggested in Biochim. Biophys. Acta 212, 362 (1970) and Anal. Biochem.33, 341 (1970). The enzyme can also be coupled through covalent bonds topolymeric macromolecules with appropriate functional groups. Thus e.g.according to Biochim. Biophys. Acta 191, 478 (1969) the enzyme isimmobilized on Sepharose 2B activated previously with cyanogen bromide,whereas according to Biochim. Biophys. Acta 377, 297 (1975) polymaleicanhydride is applied as support. According to Clin. Chim. Acta 121, 125(1980) the enzyme is immobilized on non-porous glass by carbodiimide orglutaric aldehyde coupling, whereas according to the USSR patentspecification No. 707,923 the enzyme is coupled to porous glass orsilica gel activated with cyanuric acid chloride. As described in Can.J. Biochem. 48, 1314 (1970), cholinesterase isolated from horse serumcan also be attached to a Procion brilliant orange DEAE-cellulosecomplex through covalent bonds.

A disadvantage of the known cholinesterase enzyme preparations in whichthe enzyme is entrapped in a gel is that the enzyme dissolvescontinuously from it. Therefore, such preparations are not suitable forcontinuous, prolonged application. The known cholinesterase enzymepreparations immobilized on the substrate by covalent bonds have adversethroughflow properties from the aspects of application in automaticsystems, moreover some of the supports, such as Sepharose 2B, aresensitive to microbial effects.

The invention aims at the elimination of the above disadvantages.According to the invention immobilized cholinesterase enzymepreparations are provided which meet the requirements of prolonged use,and contain the enzyme bound to a chemically and microbially inertsupport with appropriate mechanical properties ensuring a highthroughflow rate. As a further advantage, the enzyme can be coupled tothe support under mild reaction conditions.

It has been observed that polymeric resins built up from acrylic acidand/or methacrylic acid and acryl amide and/or methacryl amide monomerswith acryl or allyl type cross-linking agents [such asN,N'-methylene-bis(acrylamide), ethylene diacrylate orN,N'-diallyl-tartaric amide], containing at least 0.1 meq/g, preferably2 to 8 meq/g, of --COOH functional groups, completely meet therequirements set forth in connection with the supports. These supportscan be prepared by methods known per se. One can proceed e.g. so thatacryl amide and/or methacryl amide is copolymerized with acrylic acidand/or methacrylic acid in the presence of a cross-linking monomer,whereas according to another possible method a cross-linked polymer isprepared first from acryl amide and/or methacryl amide using the abovecross-linking agents, and the acid amide groups of the resulting polymerare subjected then to partial hydrolysis to provide the required amountof --COOH functional groups. Akrilex C type enzyme supports, marketed byReanal Finomvegyszergyar (Budapest, Hungary), prepared by subjectingAkrilex P type acryl amide --N,N'-methylene-bis(acrylamide)copolymerbeads (e.g. Akrilex P-30, P-100 or P-200) to partial hydrolysis with anacid (e.g. hydrochloric acid or another strong acid) or a base (e.g.sodium hydroxide, sodium carbonate or another strong base), belong tothis latter type of support polymers. In these substances about 50% ofthe --CONH₂ functional groups present are converted into carboxy groupsupon hydrolysis, whereas the remaining --CONH₂ groups do not hydrolyzeeven under severe reaction conditions. Consequently, unchanged --CONH₂groups are situated between the carboxy groups of the hydrolyzedcopolymer, which fix the carboxy groups in favourable steric positions.If an enzyme is coupled to the carboxy groups by a carbodiimideactivation method known per se, the functions of the individualimmobilized enzyme molecules do not interfere with each other due to thefavourable steric positions of the carboxy groups; thus the immobilizedenzyme preparation may have a very high specific activity. Polymerscontaining acrylic acid and/or methacrylic acid and acryl amide and/ormethacryl amide monomers connected to each other through a bifunctionalcross-linking monomer and carrying at least 0.1 meq/g of --COOHfunctional groups, prepared by other methods, possess similaradvantageous properties. These polymers have bead shape, thus theyenable a great throughflow rate, and at the same time they arechemically inert and completely resistant to the effects ofmicroorganisms.

The carboxy functional groups of these supports can be activated by thewell-known carbodiimide method, and the resulting activated supports canbind cholinesterase enzyme. This reaction can be performed even undermild conditions (at temperatures of 0°-4° C. in a medium of pH˜7.0).

Based on the above, the invention relates to immobilized cholinesteraseenzyme preparations which contain the cholinesterase enzyme bound to apolymeric resin, built up from acrylic acid and/or methacrylic acid andacryl amide and/or methacryl amide monomers with acryl or allyl typecross-linking agents, containing at least 0.1 meq/g of --COOH functionalgroups, previously activated with a carbodiimide.

The invention also relates to a process for the preparation of theseimmobilized enzyme preparations. According to the invention a polymericresin, built up from acrylic acid and/or methacrylic acid and acrylamide and/or methacryl amide monomers with an acryl or allyl typecross-linking agent and containing at least 0.1 meq/g of --COOHfunctional groups, is treated with a carbodiimide derivative which issoluble in water or is soluble in an organic solvent at a temperaturebelow 0° C., a solution of cholinesterase enzyme with a pH of 4.5 to 8.5is applied to the resulting activated support, the resulting product iswashed, and then dried, if desired.

Akrilex C type polymers discussed above can be applied to particularadvantage as supports for the cholinesterase enzyme.

Cholinesterase of any origin, isolated by any method, can be applied asenzyme in the process according to the invention.

To activate the polymeric support, e.g.N-cyclohexyl-N'-[β-(N-methylmorpholino)-ethyl]-carbodiimidep-toluenesulfonate or N-ethyl-N'-(3-dimethylaminopropyl)carbodiimidehydrochloride can be applied as carbodiimide derivative. It is preferredto utilize water-soluble carbodiimides for this purpose. Of thecarbodiimide derivatives soluble only in organic solvents thosesubstances can be applied in the process of the invention which haveappropriate solubility in the given organic solvent even at lowtemperatures, i.e. below 0° C.

Cholinesterase is applied to the activated support from a solution of pH4.5 to 8.5, preferably from an almost neutral solution (pH about 7.0).Cholinesterase is applied preferably as a solution formed with a 0.1molar potassium phosphate buffer (pH=7.0).

The immobilized enzyme preparation formed in this coupling reaction iswashed in a manner known per se, and then dried, if desired. The enzymepreparation is, however, also storable in aqueous suspensions at 0° to4° C.

The specific activity of the immobilized enzyme preparations accordingto the invention is 100-120 units/1 g of xerogel, which, when comparedto the activity data disclosed in Anal. Chem. 37, 1378 (1965), Anal.Biochem. 19, 587 (1967), Biochim. Biophys. Acta 212, 362 (1970) andAnal. Biochem. 51, 362 (1973), is favourable for practical use.

The immobilized enzyme preparations according to the invention are morestable in slightly alkaline media, i.e. at the optimum pH of theirapplication (pH 8.0), than the soluble enzyme itself, which is asignificant advantage in their prolonged use.

The invention is elucidated in detail by the aid of the followingnon-limiting Examples.

EXAMPLE 1

100.6 mg of Akrilex C-100 xerogel are suspended in 5.0 ml of a 0.1 molarpotassium phosphate buffer (pH 7.0), and a solution of 102.5 mg ofN-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride in 2.5 mlof a cold (+4° C.) buffer solution is added to the suspension at 0° C.under steady stirring. After 10 minutes of stirring 47.4 mg ofcholinesterase, dissolved in 2.5 ml of a cold (+4° C.) buffer solution,are added to the reaction mixture. The mixture is maintained at 0° to 4°C. for 48 hours; during this period the suspension is stirred twice for6 hours, each. The gel is filtered off, washed thrice with 10 mlportions of a 0.1 molar potassium phosphate buffer (pH 7.0), thrice with10 ml portions of a 0.1 molar potassium phosphate buffer (pH 7.0) alsocontaining 0.5 moles of sodium chloride, and then thrice with 10 mlportions of a 0.1 molar potassium phosphate buffer (pH 7.0). Thereafterthe gel is washed four times with 25 ml portions of distilled water, andthe resulting salt-free gel is subjected to freeze-drying. 106.2 mg ofan immobilized cholinesterase preparation are obtained. Activity:hydrolysis of 120 μmoles of butyryl-thiocholine iodide/min/g of drysubstance.

EXAMPLE 2

102 mg of Akrilex C-100 xerogel are suspended in 5.0 ml of a 0.1 molarpotassium phosphate buffer (pH 7.0), and a solution of 202 mg ofN-cyclohexyl-N'-[β-(N-methylmorpholino)-ethyl]-carbodiimidep-toluenesulfonate in 2.5 ml of a cold (+4° C.) buffer is added to thesuspension at 0° C. under steady stirring. After 10 minutes of stirring49 mg of cholinesterase, dissolved in 2.5 ml of a cold (+4° C.) buffersolution, are added to the reaction mixture. The mixture is maintainedat 0° to 4° C. for 48 hours; during this period the suspension isstirred twice for 6 hours, each. The gel is filtered off, washed thricewith 10 ml portions of a 0.1 molar potassium phosphate buffer (pH 7.0),thrice with 10 ml portions of a 0.1 molar potassium phosphate buffer (pH7.0) also containing 0.5 moles of sodium chloride, and then thrice with10 ml portions of a 0.1 molar potassium phosphate buffer (pH 7.0).Thereafter the gel is washed four times with 25 ml portions of distilledwater, and the resulting salt-free gel is subjected to freeze-drying.110 mg of an immobilized cholinesterase are obtained. Activity:hydrolysis of 110 μmoles of butyryl-thiocholine iodide/min/g of drysubstance.

Comparative tests and results

The activity and specific activity of the immobilized enzymepreparations according to the invention were compared to the respectivedata of cholinesterase enzyme preparations immobilized on othersupports. The following reference substances were applied:

(a) An Enzacryl AA type support (acrylamide--N,N'-methylene-bis-acrylamide copolymer containing aromatic aminefunctional groups, produced by Koch-Light Laboratories Ltd., Colnbrook,Bucks, Great Britain) was diazotized according to the prescriptions ofthe producer firm [Koch-Light Laboratories Ltd., KL 3, page 343 (1970)].A mixture of 100 mg of the resulting diazotized Enzacryl AA support and2.5 mg of cholinesterase was stirred in 0.5 ml of a 0.1 molar potassiumphosphate buffer (pH 7.5) at 0°-4° C. for 8 hours, the suspension wasallowed to stand in a refrigerator at 4° C. for 16 hours, then it wasstirred again at 0°-4° C. for 8 hours, and kept again in a refrigeratorat 4° C. for 16 hours.

(b) An Enzacryl AH type support [acryl amide-acrylichydrazide--N,N'-methylene-bis(acryl amide)copolymer, produced byKoch-Light Laboratories Ltd., Colnbrook, Bucks, Great Britain] wasactivated with sodium nitrite in acidic medium according to theprescriptions of the producer firm [Koch-Light Laboratories Ltd., KL 3,page 344 (1970)]. 100 mg of the activated support, which contains thefunctional groups in the form of acid azide groups, were admixed with2.5 mg of cholinesterase enzyme in a 0.1 molar borate buffer (pH 8.0),and the resulting suspension was stirred under the conditions describedin point (a) above.

(c) 93 mg of carboxymethyl cellulose with a binding capacity (--COOHcontent) of 2.3 meq/g, produced by Reanal Finomvegyszergyar, Budapest,Hungary, were suspended in 5 ml of a 0.1 molar potassium phosphatebuffer (pH 7.0), and 50 mg ofN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, dissolvedin 2.5 ml of a cold (+4° C.) buffer solution, were added to thesuspension under steady stirring. After 10 minutes of stirring 25 mg ofcholinesterase, dissolved in 2.5 ml of a cold (+4° C.) buffer solution,were added to the suspension. The enzyme was reacted with the activatedsupport at 0°-4° C. for 48 hours; during this time the mixture wasstirred twice for 6 hours, each.

(d) A polymaleic anhydride support produced by the firm Merck(Darmstadt, German Federal Republic) was coupled with cholinesteraseaccording to the prescriptions of the producer firm [Biochemics Merck:Poly(maleic anhydride) crosslinked enzyme support]. 1 g of the supportwas suspended in 25 ml of a 0.05 molar potassium phosphate buffer (pH7.0) at 0° C., and after one minute of stirring 0.5 g of cholinesterase,dissolved in 5 ml of a cold (+4° C.) buffer solution, were added to thesuspension. The pH of the reaction mixture was maintained between 8 and9 by adding continuously a 1 n aqueous sodium hydroxide solution to themixture. The introduction of the base was continued until the pH of themixture remained constant for about 0.5 hours.

The immobilized enzyme preparations, obtained in the reactions describedin points (a) to (d) above, were filtered off or centrifuged, and washedthen with the buffer applied at immobilization, a 0.5-1.0 molar sodiumchloride solution prepared with the same buffer, and then again with thebuffer applied at immobilization, in order to remove the proteins notbound by covalent bonds. Thereafter the ions of the buffer solution wereremoved by washing the solids with water, and the immobilized enzymepreparations were subjected to freeze-drying. The freeze-dried productswere stored in a refrigerator at 4° C.

The amounts of proteins bound in the immobilized enzyme preparationswere determined indirectly, as the difference of the amount of proteinintroduced into the reaction mixture and remaining in the supernatantand wash after immobilization. The protein content of the cholinesterasesolutions was determined by the biuret method [A. G. Gornell, C. S.Bardawill, M. M. David: J. Biol. Chem. 177, 751 (1949)].

The activity of dissolved and immobilized cholinesterases was determinedby measuring the sulfhydryl groups liberated upon the decomposition ofbutyryl-thiocholine iodide substrate. The sulfhydryl groups weremeasured with bis(5-carboxy-4-nitrophenyl)-disulfide [G. L. Ellman, K.D. Courtney, V. Andres Jr., R. M. Featherstone: Biochem. Pharmacol. 7,88 (1961)]. The amount of enzyme capable of catalyzing the conversion of1 μmole of butyryl-thiocholine iodide per minute at 25° C. and at theoptimum pH of the catalytic activity (pH 7.6 for the dissolved enzymeand 8.0 for the immobilized enzyme) was regarded as one activity unit.

The results of the tests are summarized in Table 1.

    __________________________________________________________________________                          Activity re-                                                    Immobilized                                                                          Immobilized                                                                          covered in                                                                          Loss in                                                                            Activity of                                                                         Specific                                       protein,                                                                             activity,                                                                            dissolved                                                                           activity,                                                                          the product                                                                         activity,                              Support %      %      state, %                                                                            %    *     %**                                    __________________________________________________________________________    Enzacryl AA                                                                           100    0      0     100  0     0                                      Enzacryl AH                                                                           61.8   0.2    60.7  39.1 0.2   0.3                                    Akrilex C-100                                                                         24.5   7.5    60    32.5 120   30.6                                   (Example 1)                                                                   Carboxymethyl                                                                         5.5    0.2    3.5   96.3 2     3.6                                    cellulose                                                                     Polymaleic                                                                            23.3   7.5    31.3  61.2 106   32.2                                   anhydride                                                                     __________________________________________________________________________     *unit/mg of dry substance?                                                    **the specific activity of the dissolved enzyme was regarded as 100%.    

It appears from the data of Table 1 that the preparation according tothe invention has the greatest activity related to both dry weight(protein + support) and protein content. The activity of the enzymepreparation immobilized on polymaleic anhydride support approaches thatof the new product; polymaleic anhydride is, however, a support preparedby bulk polymerization, thus the throughflow characteristics of a columnmade of cholinesterase enzyme immobilized on polymaleic anhydride aremuch poorer than those of a column made of the product according to theinvention, in which the enzyme is immobilized on polymer beads.

What we claim is:
 1. An immobilized cholinesterase enzyme preparation,in which the cholinesterase enzyme is bound to a polymeric resinactivated with a carbodiimide, said resin being built up from a firstmonomer selected from the group consisting of acrylic acid andmethacrylic acid; a second monomer selected from the group consisting ofacryl amide and methacryl amide; and a cross-linking agent selected fromthe group consisting of a compound having two acrylic moieties asterminal groups and a compound having two allyl moieties as terminalgroups; and said polymeric resin containing at least 0.1 meq/g of --COOHfunctional groups.
 2. A preparation as claimed in claim 1, in which thepolymeric resin is in the form of beads.
 3. A process for thepreparation of an immobilized cholinesterase enzyme preparation,characterized in that a polymeric resin, built up from a first monomerselected from the group consisting of acrylic acid and methacrylic acid;a second monomer selected from the group consisting of acryl amide andmethacryl amide; and a cross-linking agent selected from the groupconsisting of a compound having two acrylic moieties as terminal groupsand a compound having two allyl moieties as terminal groups; and saidpolymeric resin containing at least 0.1 meq/g of --COOH functionalgroups, is treated with a carbodiimide derivative which is soluble inwater or is soluble in an organic solvent at temperatures below 0° C., asolution of cholinesterase enzyme with a pH of 4.5 to 8.5 is applied tothe resulting activated support, and the resulting product is washed. 4.A process as claimed in claim 3, characterized in that said polymericresin contains 2 to 8 meq/g of --COOH functional groups.
 5. A process asclaimed in claim 3, characterized in that said resin is an Akrilex Cpolymer, prepared by the partial acidic or alkaline hydrolysis ofAkrilex P acryl amide--N,N'-methylene bis(acryl amide)copolymer beads.6. A process as claimed in claim 3, characterized in that cholinesteraseis applied onto the activated support as a solution in a 0.1 molarpotassium phosphate buffer (pH 7.0).
 7. A process as claimed in claim 3,characterized in that cholinesterase is reacted with the activatedsupport at 0°-4° C.
 8. A process as claimed in claim 3, in which saidresulting product is dried after said washing.